The role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence.

In recent years, frequent hexavalent chromium [Cr(VI)] pollution incidents have severely damaged the ecology and endangered the public health. It is well known that cell senescence could promote the carcinogenesis, thus the related research on the occurrence of premature senescence is of great significance to the elucidation of the carcinogenic mechanism of Cr(VI). We previously confirmed that long-term low-dose Cr(VI) exposure induced premature senescence, but the key molecular events that determine the occurrence of premature senescence are still unclear. In the present study, we found that Cr(VI) induced phosphorylation of dynamin-relatedprotein 1 (Drp1)-S637 site in premature senescent cells, which was accompanied with the decrease of mitochondrial fission. We also demonstrated that the phosphorylation status of Drp1-S637 after Cr(VI) exposure was related to the antagonism of PKA/PP2B, and continuous dephosphorylation of Drp1-S637 attenuated premature senescence caused by Cr(VI). The epidermal growth factor receptor (EGFR) overexpression significantly alleviated the occurrence of premature senescence, and the expressions of EFGR and its downstream molecules were related to the phosphorylation status of Drp1-S637. In brief, we revealed the role of PKA/PP2B-mediated Drp1 phosphorylation and the subsequent EGFR inhibition in Cr(VI)-induced premature senescence. This study is the first time to link the phosphorylation of Drp1 with Cr(VI)-induced premature senescence, in order to find the key molecular events that determine the occurrence of premature senescence and demonstrate the molecular mechanism of abnormal elongated mitochondria formation in the senescence process. The significance of this study is to explore the carcinogenesis of Cr(VI) and provide new ideas and strategies for the targeted treatment of Cr(VI)-related cancers.

Proteomic characteristics and identification of PM2.5-induced differentially expressed proteins in hepatocytes and c-Myc silenced hepatocytes.

Proteomics and bioinformatics were applied to explore PM2.5-induced differentially expressed proteins (DEPs) in hepatocytes (L02 cells) and c-Myc-silenced hepatocytes. L02 cells and c-Myc-silenced hepatocytes were treated with PM2.5 for 24 h. Fifty-two DEPs were screened in L02 hepatocytes, of which 28 were upregulated and 24 were downregulated. Forty-one DEPs were screened in the c-Myc-silenced hepatocytes, of which 31 were upregulated and 10 were downregulated. GO analysis showed that DEPs in L02 cells were mainly concentrated in the cytosol and were involved in biological processes such as the response to metal ions. DEPs in c-Myc-silenced cells were mainly enriched in the extracellular space and were involved in lipoprotein metabolism. KEGG analysis showed that DEPs in L02 cells were mainly involved in arachidonic acid metabolism and mineral absorption. DEPs in c-Myc-silenced cells were mainly enriched in pathways involving nerve absorption, complement and coagulation cascades, and other pathways. Twenty key proteins, including Metallothionein-2A (MT2A), Metallothionein-1X (MT1X), zinc transporter ZIP10 (SLC39A10) and Serine protease 23 (PRSS23) were screened in two groups through analysis of protein-protein interactions. Based on the identification of the selected DEPs, PRSS23 and SLC39A10 might be the potential biomarker of PM2.5-induced carcinogenesis, which provide the scientific basis for further research into the carcinogenic mechanisms of PM2.5.

Lipid Accumulation and IL-6 Production in L02 Hepatocytes Induced by Sodium Oleate: Dose and Time Dependence.

To explore interleukin-6 (IL-6) production and characterize lipid accumulation in L02 hepatocytes induced by sodium oleate. L02 hepatocytes were incubated with 0, 37.5, 75, 150, 300, 600, or 1,200 µmol/L sodium oleate for 24 h, and the supernatant was collected to detect the concentration of IL-6. L02 hepatocytes were incubated with 300, 150, 75, or 0 µmol/L sodium oleate for 0-24 h. The supernatant was collected for detection of IL-6 and free fatty acids. L02 hepatocytes treated with 300 µmol/L sodium oleate for 0-24 h were stained with Oil Red O. With extended sodium oleate incubation time, IL-6 levels increased, and free fatty acids decreased. After 24 h incubation, IL-6 levels increased as sodium oleate increased from 37.5 to 300 µmol/L ( P < 0.05 for 37.5 µmol/L, P < 0.01 for 75 µmol/L and P < 0.001 for concentrations 150 µmol/L or higher). Lipid accumulation increased as the sodium oleate concentration and incubation time increased. Oil Red O staining intensified with incubation time extending beyond 2 h. IL-6 production and lipid accumulation in L02 hepatocytes are influenced by sodium oleate in a dose- and time-dependent manner.

Keap1-Nrf2 pathway up-regulation via hydrogen sulfide mitigates polystyrene microplastics induced-hepatotoxic effects.

Microplastics, which are new types of environmental pollutants, are recently receiving widespread attention worldwide. Hydrogen sulfide (H2S) as the third endogenous gaseous mediator had protective effects in multiple physiological and pathological conditions. However, the protective role of H2S in microplastics-induced hepatotoxocity remain unclear. In this study, our data showed that H2S significantly suppressed inflammation, apoptosis and oxidative stress induced by polystyrene microplastics (mic-PS) (20 mg/kg b.w.) in the liver. Strikingly, although mic-PS exposure increased the expression of nuclear factor-E2-related factor (Nrf2), it did not influence the levels of heme oxygenase-1 (HO-1) and NAD(P)H: quinone oxidoreductase 1 (NQOl) in the L02 hepatocytes. Immunofluorescence assay showed that sodium hydrosulfide (NaHS) reduced micro-Ps-induced hepatic apoptosis by facilitating nuclear accumulation of Nrf2. Simultaneously, flow cytometry also showed that NaHS could prevent mic-PS-induced accumulation of reactive oxygen species (ROS) by increasing the expression of HO-1 and NQO1. Furthermore, inhibition of HO-1 could reverse the hepatic protective effects of NaHS during mic-PS exposure. Mechanistically, H2S elevating the HO-1 and NQO1 expression by facilitating nuclear accumulation of Nrf2, and consequently reducing mic-PS-induced hepatic apoptosis and inflammation. This study unveils the hepatotoxic effects of MPs and suggest NaHS have protective effects on mic-PS-induced liver damage.

Lipid Accumulation and IL-6 Production in L02 Hepatocytes Induced by Sodium Oleate: Dose and Time Dependence / 生物医学与环境科学（英文）

To explore interleukin-6 (IL-6) production and characterize lipid accumulation in L02 hepatocytes induced by sodium oleate. L02 hepatocytes were incubated with 0, 37.5, 75, 150, 300, 600, or 1,200 μmol/L sodium oleate for 24 h, and the supernatant was collected to detect the concentration of IL-6. L02 hepatocytes were incubated with 300, 150, 75, or 0 μmol/L sodium oleate for 0-24 h. The supernatant was collected for detection of IL-6 and free fatty acids. L02 hepatocytes treated with 300 μmol/L sodium oleate for 0-24 h were stained with Oil Red O. With extended sodium oleate incubation time, IL-6 levels increased, and free fatty acids decreased. After 24 h incubation, IL-6 levels increased as sodium oleate increased from 37.5 to 300 μmol/L (

Cr(VI)-induced overactive mitophagy contributes to mitochondrial loss and cytotoxicity in L02 hepatocytes.

Hexavalent chromium [Cr(VI)] has aroused the main interest of environmental health researchers due to its high toxicity. Liver is the main target organ of Cr(VI), and the purpose of this study was to explore whether mitophagy contributes to Cr(VI)-induced hepatotoxicity and to demonstrate the potential mechanisms. Cr(VI) exposure induced mitochondrial loss, energy metabolism disorders and cell apoptosis, which were associated with the occurrence of excessive mitophagy characterized by the increased number of green fluorescent protein-microtubule-associated protein light chain 3 (GFP-LC3) puncta and lysosomal colocalization with mitochondria. In addition, the suppression of mitophagy by autophagy-related 5 (ATG5) siRNA can effectively inhibit Cr(VI)-induced mitochondrial loss and cytotoxicity. In summary, we reached the conclusion that Cr(VI)-induced overactive mitophagy contributes to mitochondrial loss and cytotoxicity in L02 hepatocytes, which will further reveal the possible mechanisms of Cr(VI)-induced hepatotoxicity, and provide a new experimental basis for the study of the health hazard effects of chromium.

[Role of mitochondrial damage in cadmium-induced cell apoptosis and DNA damage of hepatocytes].

OBJECTIVE: To investigate the role of mitochondrial damage mediated by reactive oxidative species(ROS) in cadmium-induced cell apoptosis and DNA damage of L02 hepatocytes, so as to provide experimental basis for the subsequent study and protection of people exposed to Cd. METHODS: The L02 hepatocytes were cultured in vitro treated with 0-90 µmol/L Cd for 24 h, and the methylthiazolyldiphenyltetrazoliumbromide assay was used to detect the cell viability. The colony formation assay, flow cytometry, comet assay, 2', 7'-dichlorofluorescein diacetate, MitoTracker Red CMXRos and 10-N-nonyl-acridine-orange, mitochondrial membrane potential detection kit(JC-1) and adenosine triphosphate(ATP) assay kits and Western Blot were used to investigate cell growth and proliferation, cell apoptosis, DNA damage, ROS levels, mitochondrial morphology, mitochondrial membrane potential, mitochondrial mass, ATP content and related proteins after the cells exposed to 0, 20, 40 µmol/L Cd for 24 h. The cells were pretreated with vitamin C before adding Cd exposure, and ROS levels, mitochondrial function, cell apoptosis, DNA damage and proteins were measured. RESULTS: The cell viability was significantly inhibited with the increase of Cd concentration and treatment time. The cells were treated with Cd for 24 h for further study according to the result of MTT assay. Compared with control group, the colony formation rate were 8. 23% and 6. 17% respectively in 20 and 40 µmol/L Cd treatment and the apoptosis rate were 15. 85% and 26. 26%, respectively. We also found that the B cell lymphoma/leukemia(Bcl-2) gene protein was significantly reduced, while the levels of Bcl-2 associated X protein(Bax) and cleaved cysteine aspastic acid-specific protease 3(cleaved-caspase-3) were increased in a dose-dependent manner. Cd treatment also induced DNA damage and accumulation of intracellular ROS, accompanied by a mitochondrial morphological change, significant decrease in Δψm, mitochondrial mass, ATP content, mitochondrial cytochrome C(cyt c) and an increase in cytoplasmic cyt c expression(P<0. 05). In addition, pretreatment with antioxidant vitamin C not only significantly increased cyt c, mitochondrial mass, ATP content and mitochondrial cyt c, but also reduced the expression of cytoplasmic cyt c(P<0. 05), cell apoptosis and DNA damage induced by Cd. CONCLUSION: Cd exposure could induce ROS accumulation in L02 hepatocytes, which can lead to mitochondrial damage, and ultimately lead to cell apoptosis and DNA damage.

ROS-mediated miR-21-5p regulates the proliferation and apoptosis of Cr(VI)-exposed L02 hepatocytes via targeting PDCD4.

Although much has been determined about the molecular mechanisms of hexavalent chromium [Cr(VI)]-induced hepatotoxicity, more remains to be explored. In particular, explicit epigenetic alterations of microRNAs (miRNAs) which can negatively regulate mRNAs at post transcriptional level remain understudied. In the present study, cell apoptosis was determined using Annexin V/propidium iodide (PI) staining, while proliferative growth was analyzed by colony formation assay and proliferating cell nuclear antigen (PCNA) detection. miRNA microarray was performed to compare the global miRNAs expression patterns. miR-21-5p mimics (mi)/inhibitor (in), and PDCD4-siRNAs were transfected into L02 hepatocytes. Our results revealed that Cr(VI) induced apoptosis and inhibited proliferation in L02 hepatocytes via reactive oxygen species (ROS), the formation of which is closely related to mitochondrial damage, especially the inhibition of mitochondrial respiratory chain complex (MRCC). We also confirmed that ROS-mediated miR-21-5p inhibition participated in cell apoptosis and proliferative inhibition induced by Cr(VI). Furthermore, programmed cell death protein 4 (PDCD4), the up-regulation of which was related to ROS over-production, was predicted and verified as a target of miR-21-5p. Transcription factor PDCD4 silencing suppressed apoptosis and stimulated cell proliferation. In conclusion, from the perspective of epigenetics, the present study revealed that ROS-mediated miR-21-5p regulated the proliferation and apoptosis of Cr(VI)-exposed L02 hepatocytes via targeting PDCD4, which provided the new targets for molecular intervention and treatment of liver damage in Cr(VI)-exposed population.

Expression of Clusterin suppresses Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway.

Our group found that long-term low-dose exposure to hexavalent chromium [Cr(VI)] in L-02 hepatocytes resulted in premature senescence, which accompanied by the increased expression of Clusterin (CLU), but the functional role of CLU in premature senescence has never been explored. In the present study, the CLU overexpressed or silenced L-02 hepatocytes were established by lentiviral vector transfection. Cell viability assay, cell cycle analysis, western blotting, plate clone formation assay, and confocal microcopy were performed. The results indicated that Cr(VI)-induced premature senescence was associated with phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) pathway inhibition, and high expression of CLU in the senescent cells exerted its functional role of promoting cell proliferation. CLU could complex with eukaryotic translation initiation factor 3 subunit I (EIF3I) and prevent its degradation, leading to the increase of AKT activity in Cr(VI)-exposed senescent hepatocytes. Blockage of the PI3K/AKT pathway with its inhibitor LY294002 eliminated the inhibitory effect of CLU on Cr(VI)-induced premature senescence. We concluded that high expression of CLU suppressed Cr(VI)-induced premature senescence through activation of PI3K/AKT pathway, which will provide the experimental basis for the study of Cr(VI)-induced liver cancer, especially for the elucidation of the mechanism of liver cancer cells escaping from senescence.

Role of mitochondrial damage in Cr(VI)­induced endoplasmic reticulum stress in L­02 hepatocytes.

Although it is well reported that mitochondrial damage and endoplasmic reticulum (ER) stress (ERS) are involved in heavy metal­induced cytotoxicity, the role of mitochondrial damage in hexavalent chromium [Cr(VI)]­induced ERS and the correlation between the two have not been described and remain to be elucidated. The present study evaluated the ability of Cr(VI) to induce ERS in L­02 hepatocytes, and subsequently examined the role of reactive oxygen species (ROS)­mediated mitochondrial damage in Cr(VI)­induced ERS. The findings demonstrated that Cr(VI) induced ERS, which was characterized by the upregulation of ERS­associated genes and the substantial release of Ca2+ from the ER. The Cr(VI)­induced mitochondrial production of ROS, by disturbing mitochondrial respiratory chain complexes I and II, may damage mitochondria directly by inducing mitochondrial permeability transition pore opening and mitochondrial membrane potential collapse. The results additionally demonstrated that Cr(VI) induced Ca2+ release from the ER through ROS/caveolin­1/protein kinase B/inositol 1,4,5­trisphosphate receptor signaling. The application of the ROS scavenger N­acetyl­cysteine confirmed the role of ROS in Cr(VI)­mediated mitochondrial damage, ERS and apoptotic cell death. The data obtained demonstrated the role of mitochondrial damage in Cr(VI)­induced ERS and provide novel insight into the elucidation of Cr(VI)­induced cytotoxicity.

Cr(VI)-Induced Autophagy Protects L-02 Hepatocytes from Apoptosis Through the ROS-AKT-mTOR Pathway.

BACKGROUND/AIMS: Hexavalent chromium [Cr(VI)] pollution has become a global concern for both ecosystems and human health. Our previous study revealed Cr(VI) could induce both apoptosis and autophagy in L-02 hepatocytes. Here, we sought to explore the underlying mechanism of Cr(VI)-induced autophagy and its exact role in cell death. METHODS: Autophagy ultrastructure was observed under transmission electron microscope (TEM), autophagy flux was measured with double-tagged mCherry-green fluorescent protein (GFP)-microtubule-associated protein 1 light chain 3 (LC3) assay, long-lived protein degradation assay, and LC3II expression assay in the presence of lysosomal inhibitor, bafilomycin A1 (BafA1). Reactive oxygen species (ROS) level was determined using fluorescent probe dichloro-dihydrofluorescein diacetate (DCFH-DA). The expression levels of Beclin-1, LC3, p62/ SQSTM1, and AKT-mammalian target of rapamycin (mTOR) pathway-related molecules including phosphorylation (p)-AKT, AKT, p-mTOR, and mTOR were examined using real-time polymerase chain reaction (RT-PCR) and western blotting. Apoptosis was determined using Annexin V- fluorescein isothiocyanate (FITC)/propidium iodide (PI) staining. RESULTS: Our results demonstrated Cr(VI) exposure activated autophagy in L-02 hepatocytes, as evidenced by the accumulation of autophagosomes, the increase of LC3-II and degradation of p62/ SQSTM1, and the enhanced overall degradation of proteins. We also confirmed Cr(VI)-induced LC3-II elevation mainly came from autophagy induction rather than lysosomal degradation impairment. ROS-AKT-mTOR pathway was associated with Cr(VI)-induced autophagy, and ROS scavenger N-acetylcysteine (NAC) pretreatment inhibited Cr(VI)-induced autophagy by alleviating the inhibition of the AKT-mTOR pathway. Autophagy inhibitors 3-methyladenine (3-MA) and chloroquine diphosphate (CDP) promoted Cr(VI)-induced apoptotic death. CONCLUSION: These findings indicated Cr(VI)-induced autophagy protected L-02 hepatocytes from apoptosis through the ROS-AKT-mTOR pathway.

CoQ10 Deficiency May Indicate Mitochondrial Dysfunction in Cr(VI) Toxicity.

To investigate the toxic mechanism of hexavalent chromium Cr(VI) and search for an antidote for Cr(VI)-induced cytotoxicity, a study of mitochondrial dysfunction induced by Cr(VI) and cell survival by recovering mitochondrial function was performed. In the present study, we found that the gene expression of electron transfer flavoprotein dehydrogenase (ETFDH) was strongly downregulated by Cr(VI) exposure. The levels of coenzyme 10 (CoQ10) and mitochondrial biogenesis presented by mitochondrial mass and mitochondrial DNA copy number were also significantly reduced after Cr(VI) exposure. The subsequent, Cr(VI)-induced mitochondrial damage and apoptosis were characterized by reactive oxygen species (ROS) accumulation, caspase-3 and caspase-9 activation, decreased superoxide dismutase (SOD) and ATP production, increased methane dicarboxylic aldehyde (MDA) content, mitochondrial membrane depolarization and mitochondrial permeability transition pore (MPTP) opening, increased Ca2+ levels, Cyt c release, decreased Bcl-2 expression, and significantly elevated Bax expression. The Cr(VI)-induced deleterious changes were attenuated by pretreatment with CoQ10 in L-02 hepatocytes. These data suggest that Cr(VI) induces CoQ10 deficiency in L-02 hepatocytes, indicating that this deficiency may be a biomarker of mitochondrial dysfunction in Cr(VI) poisoning and that exogenous administration of CoQ10 may restore mitochondrial function and protect the liver from Cr(VI) exposure.

In vitro cytotoxicity assessment of roundup (glyphosate) in L-02 hepatocytes.

The goal of the present study was to elucidate the in vitro cytotoxicity of Roundup and to reveal the possible related mechanisms in L-02 hepatocytes. By detecting reactive oxygen species (ROS) production, glutathione (GSH)/superoxide dismutase (SOD) levels, mitochondrial permeability transition pore (PTP) open rate, apoptosis-inducing factor (AIF) release, intracellular Ca2+ concentration, and alanine aminotransferease (ALT)/aspartate aminotransferase (AST) leakage, we determined that Roundup induced anti-oxidant system inhibition, mitochondria damage, DNA damage, membrane integrity and permeability changes, and apoptosis in L-02 hepatocytes. By revealing the mechanistic insights of Roundup-induced cytotoxicity, our results are valuable for the design of preventive and therapeutic strategies for the occupational population exposed to Roundup and other pesticides.

A Ca2+ chelator ameliorates chromium (VI)-induced hepatocyte L-02 injury via down-regulation of voltage-Dependent anion channel 1 (VDAC1) expression.

Hexavalent chromium could result in cell malfunctions. Intracellular Ca2+ ([Ca2+]i) content and VDAC1 expression are both important features related to cell survial. This study aimed to explore the mechanism of cell injury induced by Cr(VI) and tentatively offer clues to repairing this cell damage using [Ca2+]i and VDAC1. L-02 hepatocytes were treated with Cr(VI)/BAPTA, and the levels of [Ca2+]i and cell injury associated with Cr(VI) were determined in addition to the effect of BAPTA. The expression of VDAC1 in Cr(VI)-induced cells was evaluated. The results showed a dose-dependent elevation of the level of VDAC1 and the mRNA level of the VDAC1 biogenesis-related gene Sam50. BAPTA could ameliorate less severe damage induced by 4µM Cr(VI) via reducing VDAC1 and Sam50. Additionally, cell injury caused by less than 4µM Cr(VI) could be ameliorated by VDAC1 knockdown. Taken together, the findings of this study suggest that inhibition of intracellular Ca2± overload could protect cells from damage and that VDAC1 plays a considerable role in Cr(VI)-induced liver injury.

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway.

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

5-Hydroxymethylfurfural protects against ER stress-induced apoptosis in GalN/TNF-α-injured L02 hepatocytes through regulating the PERK-eIF2α signaling pathway / 中国天然药物

5-Hydroxymethylfurfural (5-HMF), a water-soluble compound extracted from wine-processed Fructus corni, is a novel hepatic protectant for treating acute liver injury. The present study was designed to investigate the protective effect of 5-HMF in human L02 hepatocytes injured by D-galactosamine (GalN) and tumor necrosis factor-α (TNF-α) in vitro and to explore the underlying mechanisms of action. Our results showed that 5-HMF caused significant increase in the viability of L02 cells injured by GalN/TNF-α, in accordance with a dose-dependent decrease in apoptotic cell death confirmed by morphological and flow cytometric analyses. Based on immunofluorescence and Western blot assays, we found that GalN/TNF-α induced ER stress in the cells, as indicated by the disturbance of intracellular Ca(2+) concentration, the activation of protein kinase RNA (PKR)-like ER kinase (PERK), phosphorylation of eukaryotic initiation factor 2 alpha (eIF2α), and expression of ATF4 and CHOP proteins, which was reversed by 5-HMF pre-treatment in a dose-dependent manner. The anti-apoptotic effect of 5-HMF was further evidenced by balancing the expression of Bcl-2 family members. In addition, the knockdown of PERK suppressed the expression of phospho-PERK, phospho-eIF2α, ATF4, and CHOP, resulting in a significant decrease in cell apoptosis after the treatment with GalN/TNF-α. 5-HMF could enhance the effects of PERK knockdown, protecting the cells against the GalN/TNF-α insult. In conclusion, these findings demonstrate that 5-HMF can effectively protect GalN/TNF-α-injured L02 hepatocytes against ER stress-induced apoptosis through the regulation of the PERK-eIF2α signaling pathway, suggesting that it is a possible candidate for liver disease therapy.

EGCG attenuates pro-inflammatory cytokines and chemokines production in LPS-stimulated L02 hepatocyte.

Endotoxin lipopolysaccharide (LPS) plays an important role in the acceleration of inflammatory reaction of hepatitis as the second attack. Compounds that can prevent inflammation by targeting LPS have potential therapeutic clinical application. Epigallocatechin-3-gallate (EGCG) has potent hepatocyte-protective effect and mild anti-hepatitis virus function. Here, we investigated whether EGCG attenuated the severity of inflammatory response in LPS-stimulated L02 hepatocytes. L02 hepatocytes were pretreated with EGCG for 2 h, then stimulated by LPS at 250 ng/ml. The expression levels of chemokine regulated upon activation normal T-cell expressed and secreted (Rantes) and monocyte chemotactic protein-1 (MCP-1), pro-inflammatory cytokines tumor necrosis factor-α (TNF-α) and interferon-γ, adhesion molecule intercellular adhesion molecule-1 (ICAM-1), oxidant stress molecules nitric oxide (NO), vascular endothelial growth factor (VEGF), and matrix metalloproteinase-2 (MMP-2) were tested by enzyme-linked immunosorbent assay. The expression of total extracellular signal-regulated kinase 1/2 (ERK1/2), phospho-ERK1/2 (p-ERK1/2), p-AKT, total p38, phospho-p38 (p-p38), total p65 and phospho-p65 (p-p65), IκBα, phospho-IκBα (p-IκBα) and TNF receptor associated factor 2 were tested by western blot analysis. Our results showed that pre-treatment with EGCG could significantly reduce the production of TNF-α, Rantes, MCP-1, ICAM-1, NO, VEGF, and MMP-2 in LPS-stimulated L02 hepatocytes in a dose-dependent manner. The effect of EGCG may be related to the inhibition of nuclear factor-κB (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways by down-regulation of p-IκBα, p65, p-p65, p-p38, p-ERK1/2, and p-AKT. These results indicate that EGCG suppresses LPS-induced inflammatory response and oxidant stress and exerts its hepatocyte-protective activity partially by inhibiting NF-κB and MAPK pathways.

Suppressive Effects of Genomic Imprinted Gene PEG10 on Hydrogen Peroxide-induced Apoptosis in L02 Cells / 华中科技大学学报（医学）（英德文版）

The effects of PEG10 on hydrogen peroxide (H2O2)-induced apoptosis in human normal liver cell line L02 were investigated. The PEG10 gene was transfected into L02 cells by lipofectamine,the positive clone was screened by G418 and defined as L02/PEG10,while the cell transfected with empty expression vector (pEGFP-N1) was defined as L02/vector. L02/vector and parental L02 cells served as control. RT-PCR and Western blotting were employed to detect the expression of target genes. H2O2 (50-400 mmol/L) was administered to induce the apoptosis of L02 cells. Cells viability was measured by MTT and the morphological changes of apoptotic cells were determined by fluorescence microscopy using hoechst33342 nuclei staining. DNA fragmentation was observed by agarose gel electrophoresis. PEG10 mRNA and protein levels in L02/PEG10 ceils were significantly increased as compared with those in the control cells. After treatment with 400 mmol/L H2O2 for 24 h,the cellular growth inhibition rate of L02/PEG10 cells was significantly lower (58.2%) than that of L02 (92.5%) and L02/vector (88%). Distinct morphological changes characteristic of cell apoptosis such as karyopyknosis and conglomeration were not observed in L02/PEG10. Ladder-like DNA fragmentation in a dose-dependent manner was observed in both L02 and L02/vector cell lines,but not in L02/PEG10. PEG10 over-expression significantly inhibited cytotoxicity induced by H2O2 on human normal liver cell line L02 by antagonizing H2O2-induced apoptosis.

Protective effects of polysaccharides from Hyriopsis cumingii on hepatocytes injured by CCl_4 / 中国海洋药物

Objective To study the protective effect on CCl4-injured L-02 hepatocytes of polysaccharides from Hyriopsis cumingii(HCP) and try to explain the mechanism.Methods The L-02 hepatocytes were incubated and then injured by CCl4.The levels of AST,ALT,MDA and SOD in cultural supernatant were detected by general methods.Cell viability was assayed by MTT method.Results The polysaccharides(25,250 and 1000?g?L-1)could reduce the levels of AST,ALT,MDA in cultural supernatant which increased by CCl4.It also could boost the hepatocytes viability and elevate the level of SOD which reduced by CCl4.Conclusion The results suggest that polysaccharides from Hyriopsis cumingii have direct protective activity on hepatocytes injured in vitro.It might be associated with the anti-oxidative activity of polysaccharides from Hyriopsis cumingii.